Preparation And Preliminary Application Of Monoclonal Antibodies Against H7N9 Influenza Virus HA Protein

Through mutation and gene reassortment,influenza viruses continuously evolve and create new viruses,posing constant threats to animal and human health.In February 2013,a previously undescribed H7N9 influenza virus caused a new human influenza outbreak in the Shanghai City and Anhui Province of China.Extensive studies have been conducted to characterize this novel pathogen,and live poultry markets have been shown to play a critical role in its emergence and spread.Importantly,the H7N9virus acquired multiple basic amino acids at its hemagglutinin?HA?cleavage sites,leading to the emergence of a highly pathogenic virus in the end of 2016.To avoid the severe damage to both the poultry industry and human health posed by the newly emerged H7N9 viruses,it is imperative to develop an effective diagnostic approach.In the study,using the purified A/Pigeon/Shanghai/S1069/2013?H7N9?and A/Chicken/Guangdong/SD008/2017?H7N9?viruses as immunogens respectively,a total of 22 strains of positive hybridoma clones?2B3,2D7,4E1,5H11,1H3,1C11,1C4,4G4,1E12,1H9,2D9,2H11,15C10,11A4,1G4,1A10,4F2,14F3,8F2,13F4,7E6 and 4F3?were screened by indirect ELISA and HI methods.The antibody subclass identification results showed that monoclonal antibodies?MAbs?4E1,5H11,1H3,4G4,2H11,1G4,1A10,4F2,14F3,13F4 and 4F3 were IgG1 subclass,MAbs 2B3,2D7,1C11,1C4,4G4,1E12,1H9,2D9,8F2 and 7E6 were IgG2a subclass,MAbs 15C10 and 11A4 were IgM subclass,?chain.MAbs 2B3,2D7,1C11,1C4,1E12,1H9 and 1A10 had higher HI acitivity with the titer over7log2,while the other MAbs had low HI acitivity or no HI activity.The ELISA titers of all the MAbs ranged from 1:10³ to 1:10⁸.However,1C11 reacted with H9 subtypes nonspecific.At last,six high HI acitivity and stably anti-H7 subtype HA MAbs 2B3,2D7,1C4,1E12,1H9 and 1A10 were selected.The antigenic epitopes of HA protein of AIV were analyzed with above-mentioned six MAbs.Firstly,the antigen epitopes were screened by Pepscan technology.The analysis found that these six MAbs can not specific recognize the HA1 protein,the HA2 protein and the truncated proteins by the prokaryotic expression.Secondly,Phages displaying technology analysis showed that the MAb 1A10recognized with the consensus peptide NIEPFAW,the MAb 1A10 recognized with the consensus peptide SHRLPFT,which has no matchment with AIV HA.In addition,two MAbs?1A10 and 1C4?were selected as the primary samples for incubation,the peptide microarray analysis found that the antibodies were not specifically identified with the polypeptide sequence.However,the MAbs 1C4 and1A10 could specifically recognize HA protein by IFA.These results suggested that the epitopes of the MAbs 1A10 and 1C4 were conformational epitopes.The colloidal gold strip based on the two good biological activities MAbs?2D7 and 2B3?by pairing experiment detection.To prepare the the labeled antibody,labeling the MAb 2D7 with colloidal gold particles of 25nm;MAb 2B3 was dispensed as the test line at a concentration of 1mg/mL;Goat anti-mouse IgG was dispensed as the control line at a concentration of 1 mg/mL,the detection of H7subtype AIVs were established.Detecting the H7 subtype AIVs,the colloidal gold strip showed good broad-spectrum performance,good specificity and good sensitivity,and the dectection sensitivity with viral samples were the HA titer of 2^1/2.These results provides useful technical support for the rapid screening of H7 subtype avian influenza in the field,and meet the need of rapid early screening in the field of breeding.